Tumor-derived Syrian hamster embryo (SHE) cell lines, induced in vitro by treatment with chemical carcinogens, contained increased levels of pp60c-src kinase activity compared to preneoplastic parental cell lines and normal SHE cells. The increased kinase activity di not result from an increase in the pp60c-src content of the SHE cell lines, but represented a 4-11 fold increase in the pp60c-src kinase specificity activity. Both the extent of phosphorylation and the velocity of pp66c- src phosphotransferase activity ere increased in the tumor-derived cell lines. SHE cell lines producing chicken pp60c-src were isolated following co-transfection with plasmids bearing the chicken c-src and neor genes. Chicken pp60c-src expressed in an asbestos-transformed tumor-derived cell line showed an approximate 3-fold activation of tyrosine kinase activity compared to chicken pp60c-src expressed in the preneoplastic cell line. We suggest that these results indicate that activation of pp60c-src is mediated by trans-acting cellular factors present in the tumor-derived cells. Analysis of pp60c-src in normal SHE cells, preneoplastic cell lines and tumor-derived cell lines showed no alteration in the phosphorylation of tyr-527 or tyr-416, two tyrosine residues whose phosphorylation states have been associated with modulation of kinase activity. In addition, a strong correlation was observed between the activation of endogenous pp60c-src tyrosine kinase specific activity and the presence of additional phosphotyrosine- containing proteins. These studies indicate that the neoplastic progression of cells may be accompanied by the activation of proto- oncogene products, such as the pp60c-src tyrosine kinase, by mechanisms that may not directly involve genetic alteration of the proto-oncogene DNA sequence and that novel tyrosine phosphorylations may result from this activation.